Investigating the Molecular Mechanisms Driving 7α,25‐dihydroxycholesterol‐GPR183‐Induced Hypersensitivity

Neuropathic pain is a debilitating health concern and there is an urgent need for non-opioid analgesic targets. Our group has identified GPR183 as a novel potential therapeutic target for neuropathic pain. GPR183 is a G-protein coupled receptor that promotes the migration of immune cells in response to its ligand, 7α,25-dihydroxycholesterol (7α,25-OHC). We have shown that GPR183 is upregulated in the dorsal horn spinal cord during neuropathic pain states in rodents and intrathecal injections of 7α,25-OHC is able to induce allodynia in mice in a GPR183-dependent manner. However, the mechanism by which GPR183 activation leads to pain is unknown. These studies aim to elucidate the molecular signaling pathways that contribute to 7α,25-OHC-induced hypersensitivity. Based on previous literature, we hypothesized that GPR183 activation in the spinal cord would activate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38, leading to the production of neuroexcitatory cytokines contributing to hypersensitivity.To investigate the signaling pathways downstream of GPR183 activation in the spinal cord, we performed unbiased RNA-Sequencing following intrathecal injections of 7α,25-OHC with or without the GPR183 antagonist, SAE-14, in male and female ICR mice. We then performed follow-up studies giving pharmacological inhibitors of the identified pathways prior to intrathecal 7α,25-OHC to confirm functional relevance. We also took spinal cord tissue for western blot and enzyme-linked immunoassay (ELISA) analysis following intrathecal injections.Through our unbiased RNA-sequencing we found that many of the genes that were differentially regulated by GPR183 activation contain binding sites for NF-κB or transcription factors that are regulated by the MAPK's, ERK or p38. Additionally, we found that small molecule inhibitors of these pathways delayed 7α,25-OHC-induced mechanical allodynia. Importantly, the GPR183 antagonist, SAE-14, blocked the 7α,25-OHC-induced upregulation of these pathways, including the upregulation of neuroexcitatory cytokines interleukin-1β and tumor necrosis factor α.These findings provide novel insight into how spinal GPR183 signaling produces hypersensitivity and can contribute to neuropathic pain states.